Mutants constitutive, isolation

S. avermitilis and the biosynthesis of avermectins constitute an interesting example where traditional techniques such as chemical mutagenesis and protoplast fusion combined with recombinant DNA technology have been successfully applied in mutant isolation and strain improvement. In addition, this system offered the first opportunity to apply mutasynthesis to the production of better analogs, an application that had never before been exploited commercially. An intense doramectin development effort was therefore initiated with the Mrf-deficient mutants of S. avermitilis. The first step in this process involved the isolation of mutants deficient in 5-Omethyltransferase activity to maximize levels of the more bioactive class B avermectins [13], Thereafter, a combination of strain im-... 

D-Fucose inhibits the growth of E. coli B/r when it is added to a mineral L-arabinose medium. All resistant mutants that have been isolated and characterized were found to be constitutive mutants mapping in the araC gene [6]. 

Initiator constitutive mutants have been isolated as revertants of a strain containing deletion 719, a deletion that excises araO and all known mutant sites in the araC gene. An original set of nineteen independent revertants was isolated on mineral L-arabinose agar plates subsequent to treatment of the culture with the mutagen diethyl sulfate. The revertants were detected on the plates after approximately 6 days of incubation at 37°C [11]. r mutations have also been induced with 2-aminopurine in this deletion mutant as well as in deletion mutant 766 [14],... 

It is of interest to note that C" mutants constituted one-fourth of all the Ara mutants isolated from the wild-type strain. The significance of this will be discussed in a later section. 

Constitutive mutants (C) for the L-arabinose pathway have been isolated from the wild type as mutants resistant to the D-fucose inhibition of growth on mineral L-arabinose D-fucose medium and from D-ribulokinaseless mutants dK ) as double mutants dK O) able to grow on mineral D-arabinose medium (see Sections II,B,1 II,B,2). The former C mutants were mapped by recombination frequencies [6]. With the availability of deletions ending at various positions within the araC gene, it became possible to map the C mutant sites using deletion mapping. All the C mutations isolated as dK C were mapped by this procedure [54] (Fig. 5). 

The possibility that some of the F mutations may reside in the araB gene or that the initiator area itself contains coded information for the kinase has, however, not been entirely eliminated. Although the ratio of isomerase to kinase activity in the F was much higher than that found in the induced wild type or in C s [10], it is probable that these results can be explained on the basis of the greater instability of the kinase when present in the low concentration found in the F mutants. Employing deletion mutants 719 and 766, attempts were made to isolate heat-labile constitutive mutants. If the / site was part of the araB gene or if the F mutation could occur in this gene (and still yield an active kinase), it seemed likely that such mutations could be isolated. Several hundred Ara" revertants of these deletions were isolated at 28°C, and none were temperature-sensitive. 

The constitutive mutants so far isolated as resistant to D-fucose inhibition [6] or as revertants of D-ribulokinaseless mutants [54] all map within the araC gene as defined by araC point mutations or are closely linked to the farthest left C" point mutation at the operator end of the ara OIBAD operon (see Section VI,B, and Fig. 5). A severe test of the negative control-internal inducer model was performed with the isolation and characterization of revertants of deletion 719 that excises araO and araC [7,8] (see Section VII). Nineteen revertants analyzed were shown to map within the aral region and produced a cis-dominant constitutive phenotype I ). There were no revertants of this deletion with properties o R mutations. It has been shown in other systems lac and gal), where negative control has been established, that mutations to constitutivity in the repressor gene occur at a relatively high frequency [63,85]. 

F. Jacob, D. Perrin, C. Sanchez, and J. Monod [8]. Constitutive operator mutants isolated. 

On the other hand, it has been shown that mutants isolated on the basis of their resistance to the toxic riboflavin analog roseoflavin also exhibit a riboflavin-overproduction phenotype (Kukanova et al. 1982). Moreover, roseoflavin has been used to obtain constitutive riboflavin overproducing strains of B. subtilis (Perkins and Pcto 2002), L. lactis (Burgess et al. 2004), Lact. plantarum, Leuc. mesenteroides, and Propionibacteriumfreudenreichii (Burgess et al. 2006). Some of these have been shown to provide beneficial effects in vitamin depleted animals and could be inserted as novel starter cultures (LeBlanc et al. 2005a,b, 2006)... 

Isolation of Constitutive> Catabolite Repression, and End-Product Inhibition-Resistant Mutants... 

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